Priority is hereby claimed to EP 98204031.3 filed Nov. 30, 1998 and EP 98204203.8 filed Dec. 11, 1998.
The invention relates to artificial skin which is suitable for wound covering, and which can be used externally on various types of wounds.
Human skin forms a barrier against adverse external influences such as infections. If part of the skin is damaged, as for example in the case of a burn, complications usually arise. These complications are due to the protective function of the skin being lost, as a result of which microbial invasion may occur, and to a substantial loss of moisture that may take place at the place of the wound.
Many studies have been carried out in order to provide an artificial skin which is able to take over all, or a large part of the functions of natural skin during the period that the wound is not covered by an epidermis and dermis. To this end, the artificial skin preferably comprises autologous cells provided on a scaffold. Alternatively, the artificial skin itself may serve as carrier material for cultured autologous keratinocytes and/or fibroblasts, which have a favorable influence on the recovery of the epidermis and/or dermis. Either way, it is desired that an artificial skin, or that a scaffold material comprised by the artificial skin, has suitable properties to serve as carrier for said cells.
An artificial skin of this type must, however, satisfy various requirements. On the one hand, it should provide a barrier, so that the wound is closed to bacteria and the like, and so that substantial moisture loss is avoided. On the other hand, it must be possible for adequate water vapor transport to take place through the artificial skin. During this transport, nutrients from the underlying tissue may reach the recovering skin in sufficient quantity. Another important requirement is that the artificial skin adheres to the underlying wound bed immediately after its application to the wound. Furthermore, a permanent adhesion must be formed as a result of ingrowth of tissue.
In U.S. Pat. No. 5,147,401, an artificial skin is disclosed, of which the outer surface (the surface facing away from a wound to which the skin is to be applied) is virtually closed. This is achieved, in one embodiment, by providing a bi-layer system comprising an upper layer, which is dense and non-porous, on top of a lower layer, which is porous. In a different embodiment, this is achieved by providing a single-layer system of a segmented material, so that one of the sides of the skin is virtually closed, and the other side is fairly open.
It has now been found that when the outer surface of the artificial skin is virtually closed, a poor adherence of the skin to a wound may be observed. Under certain conditions, the artificial skin shows more or less a xe2x80x98curling-up effectxe2x80x99, in that the edges of the skin are forced away from the wound, leading to a poor adherence.
Of course, when the adherence of the artificial skin to the wound is unsatisfactory, the protection of the wound by the artificial skin is equally unsatisfactory. It is therefore an object of the present invention to provide an artificial skin which shows an improved adherence.
Surprisingly, the desired improved adherence may be obtained by the provision of an artificial skin comprising an upper and a lower side, both of which are porous. Thus, the invention relates to an artificial skin based on a copolymer of a polyalkylene glycol and an aromatic polyester, which skin has a thickness between 50 and 2000 xcexcm, and which skin has an upper and a lower side, both having a macroporosity between 10% and 95%.
An artificial skin according to the invention adheres very well to a wound when applied thereto. Under many circumstances, adherence is achieved in a period of a few minutes after application. The so-called xe2x80x98curling-up effectxe2x80x99 that has been observed with the prior art artificial skins has not been found to occur with the present skin. Furthermore, the artificial skin of the invention provides a highly suitable carrier for autologous cells, thus enabling tissue repair.
An artificial skin according to the invention is based on a specific copolymer, which is biodegradable. Advantageously, the biodegradability (the rate of degradation under certain conditions) may be controlled, depending on the envisaged site of application of the artificial skin.
The specific copolymer on which the present skin is based, is a copolymer of a polyalkylene glycol and an aromatic polyester. In a preferred embodiment, an artificial skin according to the invention is a single-layer system composed of the specific copolymer.
Preferably, the copolymer comprises 40-80 wt. %, more preferably 50-70 wt. % of the polyalkylene glycol, and 60-20 wt. %, more preferably 50-30 wt. % of the aromatic polyester. A preferred type of copolymers according to the invention is formed by the group of block copolymers.
Preferably, the polyalkylene glycol has a weight average molecular weight of from 150 to 4000, more preferably of 200 to 1500. The aromatic polyester preferably has a weight average molecular weight of from 200 to 5000, more preferably of from 250 to 4000. The weight average molecular weight of the copolymer preferably lies between 20,000 and 200,000, more preferably between 50,000 and 120,000. The weight average molecular weight may suitably be determined by gel permeation chromatography (GPC). This technique, which is known per se, may for instance be performed using tetrahydrofuran as a solvent and polystyrene as external standard.
In a preferred embodiment, the polyalkylene glycol component has units of the formula xe2x80x94OLOxe2x80x94COxe2x80x94Qxe2x80x94COxe2x80x94, wherein 0 represents oxygen, C represents carbon, L is a divalent organic radical remaining after removal of terminal hydroxyl groups from a poly(oxyalkylene)glycol, and Q is a divalent organic radical.
Preferred polyalkylene glycols are chosen from the group of polyethylene glycol, polypropylene glycol, and polybutylene glycol and copolymers thereof, such as poloxamers. A highly preferred polyalkylene glycol is polyethylene glycol.
The terms alkylene and polyalkylene generally refer to any isomeric structure, i.e. propylene comprises both 1,2-propylene and 1,3-propylene, butylene comprises 1,2-butylene, 1,3-butylene, 2,3-butylene, 1,2-isobutylene, 1,3-isobutylene and 1,4-isobutylene (tetramethylene) and similarly for higher alkylene homologues. The polyalkylene glycol component is preferably terminated with a dicarboxylic acid residue xe2x80x94COxe2x80x94Qxe2x80x94COxe2x80x94, if necessary to provide a coupling to the polyester component. Group Q may be an aromatic group having the same definition as R, or may be an aliphatic group such as ethylene, propylene, butylene and the like.
The polyester component preferably has units xe2x80x94Oxe2x80x94Exe2x80x94Oxe2x80x94COxe2x80x94Rxe2x80x94COxe2x80x94, wherein 0 represents oxygen, C represents carbon, E is a substituted or unsubstituted alkylene or oxydialkylene radical having from 2 to 8 carbon atoms, and R is a substituted or unsubstituted divalent aromatic radical.
In a preferred embodiment, the polyester is chosen from the group of polyethylene terephthalate, polypropylene terephthalate, and polybutylene terephthalate. A highly preferred polyester is polybutylene terephthalate.
The preparation of the copolymer will now be explained by way of example a polyethylene glycol/polybutylene terephthalate copolymer. Based on this description, the skilled person will be able to prepare any desired copolymer within the above described class. An alternative manner for preparing polyalkylene glycou/polyester copolymers is disclosed in U.S. Pat. No. 3,908,201.
A polyethylene glycol/polybutylene terephthalate copolymer may be synthesized from a mixture of dimethyl terephthalate, butanediol (in excess), polyethylene glycol, an antioxidant and a catalyst. The mixture is placed in a reaction vessel and heated to about 180xc2x0 C., and methanol is distilled as transesterification proceeds. During the transesterification, the ester bond with methyl is replaced with an ester bond with butylene. In this step the polyethyene glycol substantially does not react. After transesterification, the temperature is raised slowly to about 245xc2x0 C., and a vacuum (finally less than 0.1 mbar) is achieved. The excess butanediol is distilled and a prepolymer of butanediol terephthalate condenses with the polyethylene glycol to form a polyethylene/polybutylene terephthalate copolymer. A terephthalate moiety connects the polyethylene glycol units to the polybutylene terephthalate units of the copolymer and thus such copolymer also is sometimes referred to as a polyethylene glycol terephthalate/polybutylene terephthalate copolymer (PEGT/PBT copolymer).
The thickness of an artificial skin according to the invention will depend on the envisaged site of application of the skin. Generally, the thickness will be within a range of 50-2000 xcexcm. The skilled person will be able to select a suitable thickness, given a certain site of application of the skin.
An important aspect of an artificial skin according to the invention is that it has a macroporosity between 10 and 95%, on both sides of the skin. In other words, both the side of the skin facing the wound to which the skin is to be applied, and the side of the skin facing away from the wound have said macroporosity. Preferably, the macroporosity is at least 15%, more preferably at least 20%. It has been found that a skin having this specific macroporosity adheres very well to a wound.
The size of the pores in an artificial skin according to the invention preferably lies between 20 and 500 xcexcm, more preferably between 50 and 250 xcexcm.
In a preferred embodiment, particularly when before or after implantation of the artificial skin keratinocytes are applied on top of the skin, the pore size varies within the skin. Preferably, the pores at the upper side of the skin, which faces away from a wound to which the skin is to be applied, are smaller than the pores at the lower side of the skin. According to this embodiment, the chance of microorganisms finding their way through the skin into a wound, and of moisture finding its way through the skin out of the wound are very small. Moreover, the ingrowth of keratinocytes into the artificial skin is substantially prevented, while a suitable porosity of the artificial skin is still present, providing a favorable diffusion profile for nutrients and waste products.
Preferably, in accordance with this embodiment, the diameter of the pores at the upper side of the skin lies between 0.2 and 40 xcexcm, preferably between 0.4 and 2 xcexcm. The porosity at the upper side of the skin preferably is higher than 15%. The diameter of the pores at the lower side of the skin preferably lies between 20 and 300 xcexcm, more preferably between 50 and 250 xcexcm.
An important advantage of an artificial skin according to the invention is that it forms a highly suitable substrate for culturing epithelial cells,.such as keratinocytes. It has also been found to be feasible to culture fibroblasts on or within the artificial skin. Due to the porous character of the present skin, the fibroblasts can grow both on the upper and on the lower side of the skin, as well as within the skin. The keratinocytes will normally be provided on top of the skin.
If necessary, a possible delay in the initial cell growth on the present skin may be overcome by improving the cell adhesion using, for instance, laminin, collagen (type IV), proteoglycans or fibronectin, which may be applied by (pre)coating to the surface of the material which is to be covered. Improvement of the initial cell growth may further be attained by modification of the surface of the artificial skin. This modification can, inter alia, be carried out by a plasma or glow discharge treatment, radiation, monomer grafting or hydrolytic etching (e.g. with sulfuric acid).
Accordingly, the invention also relates to the therapeutic use of an artificial skin as described herein above. An important example of said use concerns the use of the skin as wound-covering material, particularly in the case of deep or large wounds, such as burns.
When the artificial skin is used per se, that is without providing it with cells prior to implantation in a patient, it may be advantageous to cover the wound to which the artificial skin has been applied with a semi-permeable membrane. This membrane ensures a suitable barrier against microbial invasion and loss of moisture until cells of the patient surrounding the wound have taken over the barrier function of said membrane, or until the membrane may be replaced by an epidermal graft or a cultivated cornified keratinocyte sheet, at which time the membrane may be removed. For a general discussion of how artificial skin may be applied to a patient, reference is made to Morgan et al., Science and Medicine, July/August 1997, pp. 6-15, Yannas, Biomedical Engineering Handbook, CRC Press, 1995, pp. 2025-2038, and Wood et al., in Essays in Biochemistry, Ed. Apps and Tipton, Portland Press, Vol. 29, 1995, pp. 65-85.
Preferably, the artificial skin is provided with autologous epithelial cells and/or fibroblasts when it is applied to a wound. These cells may be obtained from a biopsy taken from the human or animal to be treated. According to the invention, it is possible to culture these autologous cells on the artificial skin both in-vitro and in-vivo. In case the artificial skin is provided with a stratified corneum prior to implantation, the use of a semi-permeable membrane will generally not be necessary.
In case the cells are cultured on the skin in-vitro, it has been found highly advantageous when the artificial skin comprises a calcium phosphate coating. This coating facilitates the adhesion of the cells to the artificial skin. The calcium phosphate may be applied to the artificial skin by soaking the skin into a highly concentrated calcifying solution at low temperature. The calcifying solution is preferably composed of at least calcium and phosphate ions, and optionally of-magnesium, carbonate, sodium and chloride ions, which are dissolved into water by bubbling carbon dioxide gas. During the natural release of carbon dioxide gas or its exchange with air, the pH of the calcifying solution is increased and the saturation is raised until the nucleation of carbonated calcium phosphate crystals on the surface of the artificial skin. The process of bubbling and/or releasing CO2 gas through or from the calcifying solution can be repeated until a sufficient thickness of the coating has been reached.
In a preferred embodiment, the artificial skin is designed to accommodate skin glands (sebaceous, apocrine or eccrine glands), hair follicles or cells from which skin glands or hair follicles may develop. The skin glands or hair follicles may be obtained, for example, from a suitable donor, such as a cadaver, preferably having the same blood type. It is preferred, however, that autologous hair follicles are used, which may be obtained in a biopsy from the patient undergoing a skin transplantation. Cells which may be used having the ability to form or develop into hair follicles are, among others, stem cells, hair follicle dermal papilla, fibroblasts, keratinocytes, germinative matrix cells, and melanocytes. It will be understood that the invention also encompasses an artificial skin provided with skin glands, hair follicles or cells which may develop into hair follicles or skin glands.
In accordance with this embodiment, the artificial skin may for instance be provided with orifices in which hair follicles, or cells which may develop into hair follicles, may be located. These orifices may be created by drilling, possibly by laser drilling, or, and this is preferred, by, punching with a, preferably hollow, needle having the desired diameter. It is also possible to form the artificial skin in a mold provided with protuberances having the form of the desired orifices. The amount of orifices per surface unit of the artificial skin will typically depend on the density of the hair growth on the skin of the patient which is to be treated with the artificial skin. In general, there will be between 1 and 50 orifices per square centimeter. It will be clear that it is aimed at to achieve as natural an appearance of the patient""s skin after implantation of the present artificial skin as possible. The diameter of the orifices may vary, dependent on whether hair follicles or cells from which hair follicles may develop are to be provided in the artificial skin. Typically, the diameter will lie between 10 xcexcm and 10 m. The depth of the orifices will, depending on the thickness of the artificial skin, vary within a range of 5 to 2000 xcexcm, preferably of 5 to 1500 xcexcm.
An artificial skin designed to accommodate skin glands or hair follicles according to the invention may be provided with a dense top layer, preferably a semi-permeable membrane as mentioned above or an epidermal equivalent provided with keratinocytes, which may be put on top thereof, and can be kept in place by gluing it to the artificial skin using any biocompatible glue. Of course, it is also possible that the dense layer forms an integral part of the artificial skin. In fact, in accordance with this embodiment it is possible that the entire artificial skin is dense or substantially dense.
The dense top layer contributes to the provision of space and configuration for hair growth. Further, it may assist in keeping the hair in place. When the artificial skin is provided with cells which may form or develop into skin glands or hair follicles, it is also possible to apply the dense top layer on top of an artificial skin not containing orifices. In that case, a substance improving cell adhesion as discussed above, such as integrin, CD2, CD48, laminin or fibronectin, may be applied at the orifices in the artificial skin, thus defining the locations for the skin glands or hair follicles to develop. Through the orifices, cells may be seeded onto the artificial skin, which will adhere to the substance improving cell adhesion. The dense top layer may be removed before or after seeding the cells. It is another advantage of the present artificial skin that it has such an optimal degradation profile that it remains intact long enough to keep a hair, a hair follicle or a skin gland in place, while on the other hand it degrades fast enough to meet all requirements of a tissue engineered artificial skin. In addition, in case the hair follicle, the skin gland or the cells which may develop into a hair follicle or a skin gland is applied to the artificial skin when said skin is in a dry, non-swollen state, the swelling behavior of the artificial skin when in contact with an aqueous environment will greatly contribute to the keeping the hair follicle or skin gland in place.
An orifice in the artificial skin forms a chamber in which the skin gland or hair follicle may be located. Through the orifice in the dense top layer (the semi-permeable membrane), a hair may grow. Also, the dense top layer may assist in keeping the hair in place. It is one of the advantages of the artificial skin according to the invention, that nutrients will be able to reach the skin gland or hair follicle by diffusion. Alternatively, the diffusion may be amplified by providing a channel below the chamber, which is smaller in diameter than the chamber itself, and through which nutrient and waste product transport may take place.
In another preferred embodiment, the artificial skin is provided with biologically active agents, which may be released after implantation of the artificial skin in a patient. Preferred biologically active agents to be incorporated into the artificial skin are vitamins, such as vitamin C or E, antibiotics, such as gentamycin, and growth factors, such as FGF, EGF, TGF-xcex1, and IGF-1. The provision of the artificial skin with biologically active agents may be accomplished as described in EP-A-0 830 859, which is incorporated herein by reference.
Above it has been mentioned, that an artificial skin according to the invention can be applied to a wound with or without cells seeded thereon. If a cell-seeded approach is taken, the cells used will preferably be autologous epithelial cells, fibroblasts, or cells that can develop into epithelial cells or fibroblasts. Whether cells are seeded before application or whether a cell-free approach is taken, in accordance with the invention it has been found feasible to apply keratinocytes onto the artificial skin. The provision of the keratinocytes onto the skin may advantageously be carried out in one of three different manners.
In accordance with a first manner, a differentiated (stratified) keratinocyte sheet may be obtained by applying keratinocytes onto a suitable carrier which allows keratinocyte attachment (e.g. of the above described copolymer of a polyalkylene glycol and an aromatic ester) having pores which allow for nutrient and waste material transport. The pores are preferably smaller than 1 xcexcm. The keratinocytes on the carrier are preferably first cultivated submerged in a suitable culture medium, followed by cultivation at the air-liquid interphase until stratification occurs. Subsequently, the keratinocyte sheet may be detached from the carrier, e.g. using by a protease such as dispase. The thus obtained keratinocyte sheet is preferably applied to an artificial skin according to the invention with its basal side facing the artificial skin. This may be carried out either in vitro or in vivo, wherein in the latter case the artificial skin is applied onto a skin wound.
In accordance with a second manner, keratinocytes are seeded on a dense polymer film which has been provided with holes or pores for nutrient and waste material transport. The film preferably has a thickness of 5-100 xcexcm, more preferably of 10-80xcexcm, and is preferably of the above described copolymer of a polyalkylene glycol and an aromatic ester, more preferably of a copolymer of polyethylene glycol and polybutylene terephthalate. The holes or pores preferably have a diameter of less than 1 xcexcm to prevent migration of keratinocytes across the polymer film. Preferably, the holes or pores are provided in the dense film by laser drilling.
Onto this polymer film, keratinocytes may be seeded to reach a subconfluent or confluent state, at the side which, after application on an artificial skin according to the invention, faces away from the wound. Thus, the polymer film will be provided between the keratinocytes and the artificial skin. Nutrient supply to the keratinocytes may be ensured either via diffusion through the polymer film or through the holes or pores.
In accordance with a third manner, the polymer film as described in the discussion of the second manner is seeded with keratinocytes at its side which is to face an artificial skin according to the invention to which it may be applied. Thus, the keratinocytes are provided between the polymer film and the artificial skin. In one embodiment, the polymer film may be removed soon, preferably within 24 hours, after the film with the keratinocytes has been applied to the artificial skin. The keratinocytes will remain behind, attached to the artificial skin. In another embodiment, the keratinocytes are allowed to form a differentiated cornified epidermis, which leads to a detachment of the (differentiated) keratinocytes from the polymer film, which may then conveniently be removed. The formation of the cornified epidermis will usually take place after the artificial skin with keratinocytes has been applied to a wound. It is preferred that accumulating wound fluid is soaked up by an absorbent material (a dressing) which is placed on the polymer film.
The invention will now be elucidated by the following, non-restrictive example.